| Objective Hepatitis C virus (HCV) is the major cause of chronic hepatitis with a significant risk of end-stage liver cirrhosis and hepatocellular carcinoma. HCV infects an estimated 170 million persons worldwide. Its RNA genome codes for a polyprotein, which is cleaved by viral and cellular proteases to produce at least 10 mature viral protein products. HCV p7 is one of these protein. Recombinant HCV p7 could form ion channels in artificial lipid membranes. The ion transport observed in lipid membranes could be blocked by a known ion channel inhibitor, amantadine. HCV p7 is essential for infectivity of HCV. Its actual role in the virus life cycle has not been determined. We design such an experiment to explicit the molecular biological mechanisms of p7 after HCV infection. Methods 1.The recombined expression plasmid pcDNA3.1(-)-p7 was constructed, HepG2 cells were cotransfected with pcDNA3.1(-)-p7 and pCAT3-Promoter. After 48 h, cells were collected and detected for the expression of the chloramphenicol acetyltransferase (CAT) by an enzyme-linked immunosorbent assay (ELISA) kit. 2.HepG2 cells were transiently transfected with pcDNA3.1(-)-p7 , and pcDNA3.1(-) empty vector was used as control. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-p7 and pcDNA3.1(-) empty vector,respectively,and suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequence between the two groups. The obtained production was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5αin random. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. At the same time, Microarray was employed for detecting and analyzing of both mRNA from the HepG2 cells. 3. We construct the expression plasmid pCAT3-ASGPR1p, which was transfected into the hepatoblastoma cell line HepG2, then cotransfected with pcDNA3.1(-)-p7. The activity of CAT in HepG2 cells transfected was detected by a ELISA kit after 48 hours. Results 1.The activity of CAT in HepG2 cells transfected by the pcDNA3.1(-)-p7 was 87 percent lower than that of HepG2 cells transfected by control plasmid. 2.The subtractive library of genes transregulated by HCVp7 was constructed successfully. From the SSH results,it was found 15 genes were up-regulated and 6 genes were down-regulated by p7 protein of HCV. High quality mRNA and cDNA had been prepared and successful microarray screening had been conducted. From the scanning results, it was found 1 genes were up-regulated and 22 genes were down-regulated by p7. 3.The report vector pCAT3-ASGPR1p has been constructed and had been confirmed by restriction enzyme digestion and sequencing. The expression of CAT in HepG2 cells co-transfected with pCAT3-ASGPR1p and pcDNA3.1(-)-p7 was 7.7 times as high as that of pCAT3-Basic, and 2.4 as high as that of pCAT3-ASGPR1p. Conclusions The p7 protein of HCV has transregulatinging effect on SV40 promoter. The obtained sequences may be target genes transregulated by p7, among which some genes coding proteins involved in cell signal transduction, cycle regulation, translation and synthesis of protein, metabolism,apoptosis, immunoregulation, and correlated with tumor. p7 can transactivate ASGPR1 promoter, and so can up-regulate the expression of ASGPR1 gene. These may explain the possible mechanism of p7 in vivo. |
